Effect of different pH on Enzyme Activity.

EEI: 2011- Year 11 Biology

Written by: Donnet, Luke

Contents
Introduction: ...................... 3 Aim:................................... 4 Hypothesis: ........................ 4 Intro: .................................. 4 Materials: ........................... 4 Methods: ............................ 5 Results: .............................. 6 Discussion: ........................ 8 Conclusion:...................... 11 Bibliography: ................... 12

Introduction: Enzymes are protein molecules which are biological catalysts which
breakdown organic matter and lower the activation energy that is required to process the compound. The body uses catalysts to unlock the energy that is stored within the organic compounds it breaks down. There are three types of catalysts that are primarily utilized in the human body which are; amylase, protease and lipase. Each enzyme is specialized which means that on them breaks down one specific type of compound. Amylase is an enzyme that breaks down starches and into simple sugars. Amylase can be found in human saliva, this is why bread can taste sweet when you leave it on your tongue for a while because the starch is breaking down into a simple sugar. Amylase is created in the human pancreas, salivary glands and in the small intestines. Protease is responsible for breaking down protein compounds which leave amino acids (the building blocks of proteins). Lipase is the last enzyme that is used in the human body and it is responsible for the breaking down of fats and oils. (Wikipedia: Enzymes, 2011)

The primary reason why the body needs to breakdown complex compounds is for transportation. For example, protease breaks down protein compounds into amino acids. Once these compounds are at their simplest form, they are then transported to where they are needed (muscles). Once there, they are then re-synthesised back into proteins which are used to build muscles. Another reason why we need enzymes is because there are some compounds which the human body process too slowly or not at all. Enzymes speed this process of breakdown complex compounds for the body to utilize. Starch itself is not used as energy in the body; instead it needs to be broken down into a simpler compound, sugar. Starch, being a polysaccharide (meaning: a carbohydrate which is made up by a whole bunch of simpler/small carbohydrates) can be easily broken down into glucose which is a monosaccharide (meaning: a simple carbohydrate). Glucose itself can and is used in the human body as energy. (Beta Force: What are Enzymes and Why We Need Them, 2006) Each enzyme is specialized which means it can only break down one particular type of compound. Amylase is an enzyme that breaks down starch molecules only, which means that no other enzyme can break down starch and Amylase cannot breakdown any other compound. This happens because the active site of an enzyme can only support one type of substrate. (For example, amylase can only have starch as its substrate). This is because the active site of an amylase cannot support any other type of substrate other than starch molecules. A simple way to explain this method is by the key and lock concept. The key is the substrate and the lock is the active site, only one key will fit the lock. (How Stuff Works: How Cells Work Enzymes at Work, 2011)

Diagram. 1

There are three main parts to an enzyme reaction which occurs every time a complex compound is broken down; the enzyme itself, active site and the substrate. The active site of an enzyme is basically where the complex molecules are broken down into simpler compounds. The substrate is the molecule that is about to be broken down by the enzymes active site.(Biology: A Contextual Approach, 2004)

Diagram. 2

There are times when enzymes dont work like they are supposed to and when this happens, compounds called coenzymes assist the active site in breaking down its intended substrate. This happens because the active site changes its shape so the substrate is unable to properly mount onto the enzyme. It is proven that changes in the environment (such as pH and temperature) can alter the shape of the active site where it can no longer be used. When this happens, the enzyme is rendered as denatured. (Biology: A Contextual Approach, 2004)

Iodine solution is a colour indicator which detects starch molecules. The iodine indicator is a yellowish/gold which turns into a dark purple colour upon contact with starch molecules. Iodine is a heavy element which is denser than many other liquids. (lugolsiodinesolution.com: Lugol's iodine, 2007) Previous studies have found that extremes in the pH can alter the physical/chemical properties of starch. The result of this is modified starch which is commonly known as the E series. (Wikipedia : Modified Starch, 2011)

Aim: The aim of this investigation was to determine the effect on amylase activity in a
range of different pH environments.

Hypothesis: Previous studies have shown that enzymes denature more

frequently towards the extreme ends of the pH scale. Therefore the rate at which the reaction occurs will be slowed down due to the increased amount of denatured enzymes in the more extreme environments.

Intro: In this experiment, amylase solutions have been tested in confined, acidic
and basic environments, this done to simulate the enzyme activity in the human body, which can give us additional knowledge of their purpose in the human body. The data collected from this experiment could prove useful for diagnosis of patients who might be suffering from enzyme deficiencies. In this experiment, the independent variables will be the changes in the pH at which the reaction occurs and the dependent variable will be the colour changes during the pH test. The amount flour put into the test tubes are the independent variables, the colour and turbidity changes are the dependent variables in the pre-tests. The volume of the solution, temperature, amount of amylase solution added and the shape of the test tubes are kept as constant as possible.

Materials:
Material/Equipment
9 test tubes 100ml of 2% amylase solution 500g of flour 300ml of 8mol hydrochloric acid 300ml of 2mol hydrochloric

Justification
A place for all the reactions occur Its the enzyme used to conduct this experiment A source of starch for the enzyme to react with The strong acid used to conduct experiment A weaker acid used in this

acid 500ml of water 300ml of 8mol of sodium hydroxide 300ml of 2mol of sodium hydroxide Iodine solution 1ml liquid dropper

Spatula Safety goggles Test tube rack Beaker

experiment. The neutral/controlled that was used in the experiment. The strong base chemical that is used in the experiment The weaker base that is used in the experiment A starch colour indicator Used to accurately drop the same amount of solution in every time Used to put power in the test tube Protect eyes from chemicals Hold the test tubes through the duration of the experiment Used as a safe way to decant liquid from the bottles into the test tubes.

Methods:
Conducting Pre-tests Step 1. Put four test tubes in a single file on the test tube rack Step 2. From left to right, mark each of the test tubes as follows; 0.5, 1, 1.5, 2. Arrange them in ascending order on the test tube rack. Step 3. Put the amount of flour that is displayed on the test tube from Step 2. (ie, the test tube marked 0.5 should have 0.5 grams of flour inside it) Step 4. Put 10ml of water into each test tube using a measuring cylinder. Step 5. Squirt 1ml of 2% amylase solution using a pipette. Step 6. Put 5 drops of iodine solution into each test tube. Step 7. Shake the 4 test tubes and record any colour changes. (results best presented in a table)

Conducting Test Step 1. Put five test tubes in a single file in the test tube rack. Step 2. Label the test tubes from left to right (S 8m, S 2m, N, H 2m, H 8m) Step 3. Using the spatula and electronic scales, accurately measure 0.5 grams of flour into each test tube. Step 4. Put; 5 5ml of 8mol Sodium Hydroxide into the test tube marked S 8m 5ml of 2mol Sodium Hydroxide into the test tube marked S 2m

5ml of water into the test tube marked N 5ml of 2mol Hydrochloric Acid into the test tube marked H 2m 5ml of 8mol Hydrochloric Acid into the test tube marked H 8m

Step 5. Shake the test tubes to get as much flour suspended in the solutions as possible. Step 6. Quickly add 1ml of 2% amylase solution into each of the test tubes. Step 7. Add 5 drops of iodine solution into each of the test tubes. Step 8. Record observations (looking for any colour changes)

Results:
During the pre-test, the changes in colour and turbidity give a clear indication of enzyme activity when added to assorted concentrations of flour. Pre-test:
Test Tube Amount Of Flour One Two Three Four Five 0.5g 1g 1.5g 2g 2.5g Light Purple Purple Deep Purple Deep Purple Deep Purple Initial Colour Colour After 10 minutes Clear/Transparent Clear/Transparent Light Purple Deep Purple Deep Purple Very Low Low Medium Very High Very High Table. 1 Turbidity

Test: Colour changes of solution when amylase activity is exposed to different levels of pH.
Chemical Permanent Colour Change
8mol Sodium Hydroxide (NaHO)

Initial Colour Change

Table. 2 No Colour Change

2mol Sodium Hydroxide (NaOH)

Water (H2O)

2mol Hydrochloric Acid (HCl)

2mol Hydrochloric Acid (HCl)

Observations: Pre-test: Test tube one with the 0.5 grams initially turned a light/transparent purple colour and after a couple of minutes, the solution turned clear but still a little turbid (semi-transparent). Test tube two with 1g of flour initially turned a soft purple colour and after a few minutes the solution turned clear but still a little turbid (semi-transparent). Test tube three with 1.5g of flour initially turned a dark solid purple colour and after a few minutes it changed to a lighter purple colour which was moderately turbid. (hazy). Test tube four had 2 grams of flour which initially turned an extremely deep, dark purple colour. After a few minutes the solution still was a dark purple colour which had a very high turbidity (opaque). Test tube five had 2.5 grams of flour initially turned an extremely deep dark purple colour. After a few minutes the solution was still a dark purple with high turbidity (opaque). In test tubes four and five, there was never a time where all the flour was suspended in the water. In all of the test tubes, after the 10 minutes was over there was at least 70% of the original mass of the flour resting at the bottom of each test tube. After the 10 minutes in test tube 1, there was a thin layer of purple coloured flour resting at the bottom of the test tube. Almost immediate reaction when the iodine solution was added.

Enzyme Test: The test tube which contained water immediately turned purple.

The test tubes which contained the Hydrochloric Acid turned a dark colour as soon as the iodine solution. As the iodine solution was added to the acids, their turbidity increased dramatically, there was no light that was able to make it through the solution. The test tubes which contained the bases had no colour at all throughout the duration of the experiment. The bottom 20% of the basic solutions had a yellowish tinge. After 10 minutes, the acids were still a dark purple/black colour. It had no significant change since the iodine was added initially. After 10 minutes, the test tube which contained water had a much lighter colour than initially. In both of the test tubes which contained basic chemicals (NaOH), the turbidity of the mixture increased approaching the bottom of the test tube.

Discussion:
Analysis:
Diagram. 3

Green = Base Blue = Water Red = Acid

The image above shows the success rate amylase had when breaking down the starch compounds. This pie graph also displays the trends and patterns of results where; initial change is when the solution went from an initial purple to a more transparent colour, No colour change is when the solution didnt change colour at all and permanent colour change is when the solution turned purple and remained purple without and colour change. These results were compared to the controlled pre-tests which were displayed in table.1 which displays the severity of the changes as well.

The results show that test tube which contained the neutral substance (water) enabled the enzymes (amylase) to work the most efficiently. This is because there was nothing that caused the denaturing of the enzyme, as a result of this the process of breaking complex molecules (starch) was able to be carried out as per normal which matched up with the control tests. 8 tests were conducted in exactly the same manner (to minimize any introduced variables) which presented identical results for each test. When starch comes into contract with a strong base such as sodium hydroxide, its chemical properties change and it turns into a carbohydrate commonly known as Alkaline-Modified Starch. This means that colour indicators which are supposed to detect the presence of generic starch will be unable to detect the presence the new modified starch. Since there was no colour change at all, assumptions can be made that the sodium hydroxide changed the properties of the starch turning into AlkalineModified Starch. This means it was impossible to track the progress of amylase breaking down any starch particles. Also, Iodine solution must have stained the flour while most of it was suspended in the water. This explained the yellow tinge that was at the bottom of the test tubes which contained the sodium hydroxide. When starch comes into contract with hydrochloric acid, a carbohydrate called Dextrin is produced. Dextrin is a form of modified starch but unlike the AlkalineModified Starch, Dextrin is indicated by iodine solution. This explains why the test tubes containing hydrochloric acid reacted while the test tubes containing strong bases didnt. The hydrochloric acid prematurely broke down the starch molecules which essentially unlocked the Dextrin from the flour grains, this explains why the acids had an extremely dark colour due to the abundance of Dextrin molecules. But since the colour never went clear, it is safe to assume that the low pH must have denatured the amylase enzymes. (Wikipedia : Modified Starch, 2011)

Variables: The amount of liquid needed to be controlled because any difference in the amount of space that was available for the reaction takes place could have affected the rate of the reaction. During the pre-tests there was a constant 10ml of water which was added into each test tube and during the real testing there was a constant 5mls of chemical which was added into each test tube. Because the pre-tests were done to provide some insight into what I should find, the volumes didnt need to be the same.
Diagram. 4

The previous diagram shows how the amount of space there is available influences the rate of the reaction just so long as the amount of amylase and starch in the test tube remains constant. The more concentrated the solution is, the more contact the more chance a substrate with attach itself to an active site. Which means the reaction will occur faster than if the mixture of amylase and starch was less concentrated. Therefore the amount of flour and amylase that was added into each test tube needed to be kept constant to have accurate results. (Yahoo Answers: How does the amount of enzyme effect enzyme reaction rate?) Also the temperature was kept constant throughout all of the testing samples to ensure that heat was not a complicating factor in any of the reactions which need to be taken into account when compiling data. The independent variable was the difference of pH in the different test tubes, it was the only thing that was changed throughout the duration of the experiment. The reason for this was to ensure than there were no other factors involved in the rate at which the amylase breaks down the starch. The dependent variable was the colour change in this experiment, the controls showed what a successful test looks like. The colour change was the sign that enzymes were doing their job correctly. Since the rate at which the enzymes work is shown by the colour change, and the rate the enzymes work is influenced by the pH in its environment. The colour change is dependent on the pH (the independent variable) in the environment which surrounds the enzyme (eg. Sodium Hydroxide, Water and Hydrochloric Acid). Suggestions for improvement: The main problem with the experiment which was never addressed was that the bases and acids were too strong (Sodium Hydroxide has a pH of 14 and Hydrochloric Acid has a pH of 0). This would have denatured the amylase almost instantaneously upon contact, which left them no time for the reaction to take place. It would have been better to use weaker bases and weaker acids or even to test in more pH environments to get a wider spread of data. Previous studies have proven that amylase from human saliva is most efficiently between a pH of 5 and 9 (the table below displays the results from the research). (Reactions and Enzymes, 2010)

Diagram. 5

The table adjacent represents enzyme activity vs pH. Taken from : http://www.emc.maricopa.edu/faculty/farabee/BIOBK/biob ookenzym.html (accessed 15/9/2011)

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Another problem which was encountered during the experiment was the fact that flour is not a water soluble substance. This means that the flour grains stay suspended in the water for a short amount of time. A suggestion to improve this would be to use a source of starch where each grain is much finer, such as rice water. Also, to maximize the amount of flour suspended, the method for getting the flour suspended could also be changed.

Diagram. 6

The image above shows that turning the test tube on an angle will increase the surface area of the flour that comes into contact with the chemical that is poured. Therefore there will be more starch suspended in the liquid.

Conclusion:
The purpose of this investigation was to determine the effects of different pH on amylase enzymes. Compared to the control, there was evidence that the pH of the solution in the test tube did have a major influence in the rate in which the reaction occurred. It was proven that extremes in the pH slow the rate the amylase breaks down starch. This was due to the potency of the chemicals being used which resulted in whole lot of complicating factors which were introduced into the experimental side of the investigation which means the original hypothesis was neither accepted nor denied. Although each test tube showed difference compared to each other and the results were consistent though out the experiment, there was not enough evidence given by the experiment to propose a valid conclusion to the experiment. Further testing with a wider range of data is required to completely determine the differences in the rates of reaction in different pH in an environment. The data gathered from this experiment could have real life implications for patients who have enzyme deficiencies. For example, a patient who is experiencing low energy levels might have an amylase deficiency from not having enough carbohydrates being broken down in the body. Amylase deficiency also related to other complications such as inflammation, allergic reactions, eczema, psoriasis and atopic dermatitis. (Buzzle: Amylase Levels in the Blood, updated 2011)

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Bibliography:
Wikipedia, Unknown Author. 2011. http://en.wikipedia.org/wiki/Enzyme [accessed 30/7/2011] This Wikipedia article introduces the basics about enzymes and their role in the human body. The aim for the article was to provide the reader with a basic knowledge of biological catalysts and the author uses results found in other studies and images to entice and help the reader understand. The scope of article was only to provide a basis of what is and how enzymes work. This article was extremely useful to my research because it provided enough information about enzymes for me to perform the experiment and form a suitable hypothesis. The only limitation to this article was the fact that most of the information that was given was irrelevant to my investigation. This article provided an adequate amount of information to complete the understanding of how enzymes work, more research is required to show how they react when exposed to different conditions. Wikipedia:Enzymes formed the basis of the investigation.

Beta Force: What are Enzymes and Why We Need Them, Unknown Author. 2006. http://www.beta-glucan-info.com/enzyme_facts.htm [accessed 30/07/2011] This internet article focuses purely on what enzymes do in our body. The author uses simple theories and explanations to all for more accessibility to the article. The purpose of this article was to inform the on basics on how enzymes work in the human body. This article was useful to the investigation because it gave enough information about the activity of enzymes to tie in with the aim of then experiment. The only limitation to this article was that it might have been too brief and the information can be found in greater depth and technicality on other sites. Although this article provides only a small portion of the understanding, it was a necessary part of the research.

Brain, Marshall. "How Cells Work" 01 April 2000. HowStuffWorks.com. <http://science.howstuffworks.com/environmental/life/cellular-microscopic/cell.htm> [accessed 1/08/2011.] This article is basically a small glossary which contained anything relevant to enzymes. The author doesnt introduce any complex ideas throughout the whole article, only defines certain terminology. The purpose of this article was to get a quick idea of what something means (such as inhibitors)

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The major limitation to this article what that there was very little actual information on the processes of enzyme reactions. Although it did not make up the bulk of the research, it provided a good quality reference point for definitions.

Spenceley M, Weller, B. Mason, M, Fullerton, K. Tsilemanis, C. Evans, B. Ladiges, P. McKenzie, J. Batterham,P. Pearson Education Australia, 2004. Rate of Enzyme Reaction Heinemann Queensland Science Project Biology: A Contextual Approach, Melbourne., pp 168-169. This text book was created to give senior school students a vivid understanding of all the aspects in biology. The authors kept in mind the intended audience for this text book, so they used both complex and simple explanations for theories as well as images and diagrams to increase the ease of learning a new concept or idea. This book was extremely useful as it provided information about all the aspects of this investigation and the sources are trustworthy. The only limitation to this book was that there was nothing that explained anything about the chemistry side of enzymes. Although nothing was taken out and used in the introduction, it did serve as a good reference point for everything in the introduction.

Other References:

Unknown Author. 2007, http://lugolsiodinesolution.com/ [accessed 25/8/2011] Unknown Author. 2008, http://answers.yahoo.com/question/index?qid=20080914115624AADqZSQ [accessed 25/8/2011] Sandhyarani.N. 2011, http://www.buzzle.com/articles/amylase-levels-in-blood.html [accessed 26/8/2011] Unknown Author. 2010, http://www.emc.maricopa.edu/faculty/farabee/BIOBK/biobookenzym.html [accessed 26/8/2011]

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